pH-dependent fibrillogenesis of a VkappaIII Bence Jones protein.

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma-cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light-chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8-5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VkappaIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post-mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N-terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril-formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro-environments may contribute to the stabilization of amyloidogenic conformations.

IgG1-kappa biclonal gammopathy associated with multiple myeloma suggests a regulatory mechanism.

Multiclonal gammopathies associated with multiple myeloma may result either from a neoplastic transformation of a cell clone undergoing immunoglobulin class switching or from independent transforming events yielding proliferation of unrelated plasma cell clones. The simultaneous presence of more than one neoplastic clone may possess regulatory implications in terms of cell proliferation, clonal expansion, secretion of M-components or response to chemotherapy. We report a patient, diagnosed with multiple myeloma stage IIIa, who presented with two well-defined homogeneous IgG1-kappa components in the serum (designated WER-1 and WER-2) with striking differences in their plasma concentration and response to the classic melphalan/prednisone treatment. Immunochemical characterization and amino terminal sequence analysis of both the heavy and light chains of each M-component undoubtedly determined their biclonal origin. WER-1 was identified as IgG1(VHII)-kappaI while WER-2 was classified as IgG1(VHIII)-kappaIII. The plateau phase was characterized by very low or undetectable levels of WER-2, a high, almost constant, concentration of WER-1 and the absence of Bence Jones proteinuria, whereas these parameters were completely reversed during the escape phase with levels resembling those observed at the time of diagnosis. The statistically significant negative correlation between the biclonal components and the different susceptibility to the treatment clearly suggests regulatory interactions between the clones WER-1 and WER-2.

Alzheimer's soluble amyloid beta is a normal component of human urine.

Soluble A beta (Sa beta) is normally present at a low concentration in human plasma and cerebrospinal fluid. Although the factors involved in the regulation of Sa beta plasma levels are still unknown, we have explored its excretion in the urine as one of the possible homeostatic mechanisms. The presence of Sa beta in the urine was investigated via immunoprecipitation experiments with anti-A beta antibodies followed by detection and identification by immunoblot, MALDI mass spectrometry and sequence analysis. Soluble A beta (4.3 kDa) immunoreactivity was present in the urine of normal donors, Down's syndrome individuals as well as in patients with renal disorders exhibiting glomerular or mixed proteinuria. Edman degradation of the immunoprecipitated material yielded the intact A beta N-terminus and mass spectra analysis indicated the existence of a major component at mlz 4327, corresponding to the molecular mass of A beta1-40. Semiquantitative data obtained from the immunoprecipitation experiments indicate that under normal conditions the daily excretion of intact Sa beta in the urine represents less than 1% of the circulating pool.

Fibrillary glomerulonephritis related to serum fibrillar immunoglobulin-fibronectin complexes.

Fibrillary glomerulonephritis is a disease of uncertain origin and pathogenesis characterized by nonamyloidotic fibrils in glomeruli. We report immunohistological, immunochemical, and biochemical studies of a serum fibrillar cryoprecipitate obtained from a patient with fibrillary glomerulonephritis, that formed on prolonged storage at 4 degrees C. By Western blot and amino acid sequence analysis, the cryoprecipitated fibril components consisted of immunoglobulins, heavy chains gamma and mu, light chains kappa and lambda, and fibronectin, similar to the proteins identified by immunofluorescence and immunoelectron microscopy in the glomerular fibrils. These findings support the hypothesis that serum precursors may be the source of the fibrillar deposits and suggest a role for immunoglobulin-fibronectin complexes in the pathogenesis of fibrillary glomerulonephritis.

Susceptibility of monoclonal IgG paraproteins to plasmic cleavage using glycerin-stabilized human plasmin.

To shed further light on plasmin-IgG interactions a simple procedure is described that permitted 48 monoclonal IgG isolates from human serum to be profiled for their susceptibility to plasmic cleavage. In addition to anodal Fc and cathodal Fab fragments, combined immunoelectrophoresis-electrophoresis revealed transient anodal banding, as well as Fab-fragment subcleavage in many of the IgG subclass-1 isolates. The subcleavage of released Fab fragments, which bear the idiotype determinants, points to a possible ancillary role of plasmin in "idiotype processing" leading to immunoregulatory anti-idiotype networks. The cleavage of IgG by endogenous plasmin also points to a possible active role of plasmin in the "steady-state" metabolism of IgG.

A paraneoplastic mixed bullous skin disease associated with anti-skin antibodies and a B-cell lymphoma.

BACKGROUND: The full spectrum of bullous diseases associated with underlying cancers remains to be fully defined. OBSERVATION: We describe a patient with a mixed bullous disease exhibiting combined features of cicatricial pemphigoid and pemphigus and associated with a B-cell lymphoma producing an IgM paraprotein to intercellular antigens in human skin. The patient had the clinical features of cicatricial pemphigoid and the histologic and immunofluorescence abnormalities of both cicatricial pemphigoid and pemphigus. These included oral and cutaneous erosions; ocular scarring; subbasal and acantholytic intraepidermal bullae; and circulating and tissue-fixed basement membrane zone and intercellular antibodies. The antibodies were directed to a 140-kd antigen in dermal extracts of skin split with 1 mol/L of sodium chloride and to antigens with approximate molecular weights of 150, 180, 230, and 285 kd in the dermal extract. In contrast to paraneoplastic pemphigus, the intercellular antibodies did not react to mammalian bladder. The intercellular antibodies were of the IgM class and were associated with the paraprotein produced by the malignant B cells. CONCLUSIONS: We believe that this condition represents a novel bullous disease, which we refer to as paraneoplastic mixed bullous disease. This condition illustrates that distinct bullous diseases are associated with paraneoplastic syndromes and that at least one possible mechanism for such eruptions is the production of anti-skin antibodies by malignant B cells.

Glucose in epidural catheter aspirate.

Frequently fluid may be aspirated from epidural catheters during epidural anesthesia/analgesia. This fluid may be either cerebrospinal fluid or local anesthetic. Several methods for differentiation of the two fluids have been recommended. In this study, the reliability of the "glucose test" was analyzed. Epidural catheters were inserted into 43 healthy, nondiabetic parturients and then gently aspirated. Aspirate was tested for glucose using glucose oxidase paper. Positive aspirates were assessed for the presence of prealbumin, which is a protein marker for cerebrospinal fluid. Of the patients undergoing cesarean section, 17 of 27 patients yielded a glucose-positive aspirate, and of the patients undergoing labor analgesia, 6 of 16 patients were found to be positive for glucose. None of these patients developed total spinal anesthesia or postdural puncture headache. Visually, none of the aspirates were significantly blood-tinged, blood being a possible source of glucose. When the glucose-positive aspirates were subjected to immunoelectrophoresis, 6 of 7 aspirates revealed a prealbumin band. In conclusion, the glucose test for cerebrospinal fluid may be misleading. The source of this glucose may be normal cerebrospinal fluid drainage into the epidural space.

Monoclonal gammopathy of undetermined significance: a morphologic and immunophenotypic study of the bone marrow.

A monoclonal paraprotein in the serum or urine raises the possibility of myeloma. However, in a significant proportion of individuals with serum paraproteins, particularly those with low levels of paraprotein, clinical and routine bone marrow evaluation is not diagnostic of an underlying neoplasm. The purpose of this study was to define the pathologic basis for monoclonal gammopathy in patients whose bone marrow biopsies showed no evidence of myeloma. We used immunofluorescence microscopy and flow cytometry of cell suspensions prepared from aspirated marrow, as well as immunohistochemistry of core biopsies, to perform immunopathologic evaluations of the bone marrow from 26 such patients. Eighteen patients with myeloma and seven without a serum paraprotein or evidence of myeloma were similarly studied. The data indicate that 17 of the 26 patients with monoclonal paraproteins whose routine bone marrow biopsies were normal or nondiagnostic had, in fact, a dispersed monotypic plasma cell population of concordant immunoglobulin heavy and light chain type in the bone marrow demonstrable by at least one of the three analytic methods. Among these, immunofluorescence microscopy of isolated bone marrow mononuclear cells was the most sensitive assay. Immunophenotypic evaluation of the bone marrow is useful for documenting and quantifying a monoclonal plasma cell population in patients with monoclonal gammopathy of undetermined significance.

IgM monoclonal gammopathy/Waldenström's macroglobulinemia: a morphological and immunophenotypic study of the bone marrow.

The presence of a monoclonal paraprotein in the serum or urine raises the possibility of myeloma or lymphoma/leukemia. Yet, in a significant proportion of individuals with serum paraproteins, particularly those with low levels of paraprotein, clinical and routine bone marrow evaluation is not diagnostic of an underlying neoplasm. The purpose of this study was to define the pathologic basis for macroglobulinemia in patients whose routine bone marrow biopsies were not diagnostic of a lymphoplasmacytic neoplasm. We used immunofluorescence microscopy and flow cytometry of cell suspensions prepared from aspirated marrow, as well as immunohistochemistry of core biopsies, to perform immunopathologic evaluations of the bone marrow from 16 such patients. Seven individuals without a monoclonal serum paraprotein, who were similarly studied, served as controls. Our data indicate that 13 of the 16 patients with monoclonal serum IgM paraproteins whose routine bone marrow biopsies were normal or showed nondiagnostic changes morphologically had a dispersed monotypic B lineage population of concordant immunoglobulin heavy and light chain type in the bone marrow. The immunophenotype of these cells spanned the range from mature B cell to plasmacytoid B cell to plasma cell. In four of these 13 patients a diagnosis of lymphoplasmacytic lymphoma could be made on the basis of greater than or equal to 20% monoclonal B lineage cells among bone marrow mononuclear cells.

Monoclonal immunoglobulin deposition disease: light chain and light and heavy chain deposition diseases and their relation to light chain amyloidosis. Clinical features, immunopathology, and molecular analysis.

Monoclonal immunoglobulin deposition occurs in tissues as Congo Red binding fibrils in light chain amyloidosis, as less structured deposits in light chain deposition disease, and as similar but distinct deposits in light and heavy chain deposition disease. The nonamyloid forms were found in 13 patients who had evidence of plasmacytic dyscrasia by the immunohistochemical detection of immunoglobulin light chains of kappa or lambda class (with or without staining for a single heavy chain isotype) and by the absence of amyloid P component in tissue sections that did not show the birefringence characteristic of amyloid after Congo Red staining. All but two of the patients presented with proteinuria with or without azotemia. Clinical syndromes involving other organ systems were less common but occasionally severe. Four patients had overt multiple myeloma. Three others had hypercalcemia and mild bone marrow plasmacytosis but no lytic lesions. Analyses of immunoglobulin synthesis in bone marrow cells from seven patients showed excess light chains in all and incomplete light chains or heavy chain fragments in six, regardless of whether an intact monoclonal protein or related subunit was in the serum or urine. The fibrillar (amyloidotic) and nonfibrillar forms of monoclonal immunoglobulin deposition occur either in overt multiple myeloma or in the course of less neoplastically aggressive plasmacytic dyscrasias. Bone marrow cells from patients with either type produce immunoglobulin fragments that are related to those deposited in the affected tissues.

A double monoclonal IgG1 kappa and IgG2 kappa in a single myeloma patient. Variation in clonal products and therapeutic responses.

Two electrophoretically homogeneous immunoglobulins were detected in the serum of a patient with multiple myeloma. The heavy chains were of different classes (gamma 1 and gamma 2). The light chains of both were kappa, but had different electrophoretic mobilities on polyacrylamide gels. Amino acid sequence analysis, carbohydrate determinations, and biosynthetic experiments indicated that the difference seen in their electrophoretic mobility was due to the glycosylation of one but not the other kappa-chain. The primary structure of both chains demonstrated that they both used a V kappa 1 and a J kappa 2 gene segment and the same C kappa allele, Km(1,2), and that both contained the same junctional three amino acid deletion. However, they varied by 19 amino acids in the first 94 amino terminal residues encoded for by the V kappa gene, with some of the substitutions requiring two base changes in the appropriate codons. Moreover, the malignant "clones" producing the two proteins differed in their responses to chemotherapy. These data indicate that, although the two clones producing the serum proteins were different at the time of study, they may have arisen from the same precursor clone.

In vitro magnetic resonance properties of CNS neoplasms and associated cysts.

Fresh surgical specimens of central nervous system (CNS) neoplasms were analyzed with particular attention to differences between the T1 and T2 values of the solid and cystic components. Delineation of solid tumor from cyst is important, particularly when surgical intervention is planned, since only the solid portion need be excised. Total protein concentration determinations and microimmunoelectrophoresis for protein distribution and characterization also were performed on the fluid specimens. To diagnose a lesion on magnetic resonance based on T1 and T2 measurements, one must first have a catalog of values on which to base that diagnosis. The authors are reporting such values at 0.25 T. In addition, protein analysis of the fluid specimens has shown that the cysts of the CNS associated with CNS neoplasms are, in fact, transudates rather than collections of cerebrospinal fluid (CSF). Their T1 should permit differentiation from solid portions of neoplasms and from non-neoplastic syringohydromyelia.

Characterization of tissue amyloid by immunofluorescence microscopy.

Immunohistochemical classification of amyloid type was possible in 44 of 50 (88%) patients as judged by the concordance of immunofluorescence, clinical, serum, and urine immunoelectrophoresis, and bone marrow data. In frozen tissue sections incubated with a panel of antisera monospecific for immunoglobulin heavy chains, kappa and lambda light chains, and amyloid-A-related protein, the amyloid was classified as AL in 20 and AA in 24. In 6 patients the amyloid could not be classified because of the absence of reactivity in 2 and overlap staining in 4. The findings indicate that routine immunofluorescence examination of diagnostic biopsies is an important adjunct in the classification of amyloid.

Transfer of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 across the perfused human placenta.

The rates of placental transfer of 25-hydroxyvitamin D3 [25-(OH)D3] and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were determined in an in vitro perfusion system. Antipyrine was included in each perfusion, and the data are expressed as clearance index, the ratio of hydroxyvitamin D3 to antipyrine clearance. In most experiments, serum, 0.2%, was added to the perfusates as a source of vitamin D3-binding protein. Binding as measured by dextran-coated charcoal assay for 25-(OH)D3 was over 90%, for 1,25-(OH)2D3, only 25% to 50%. The clearance index from the maternal to fetal circulation averaged 0.02 and 0.26 for 25-(OH)D3 and 1,25-(OH)2D3, respectively. When vitamin D3-binding protein was omitted from the perfusate, the clearance indices of 25-(OH)D3 were 0.12 and 0.46 in two experiments. Binding to vitamin D3-binding protein is a major determining factor for the transfer rates of 25-(OH)D3 and 1,25-(OH)2D3.

Nodular glomerulopathy associated with nonamyloidotic kappa light chain deposits and excess immunoglobulin light chain synthesis.

A nodular glomerulopathy characterized by mesangial deposits of monoclonal kappa light chains was detected by immunofluorescence in a renal biopsy from a patient with proteinuria and hypertension. These nodules lacked the tinctorial and morphologic features of amyloid. Ultrastructurally, the nodules contained electron-dense granular deposits as well as fibrils in parallel arrangement. The fibrils measured 110-140 A in diameter. They were consistent in size with amyloid fibrils. However, they differed in lacking the randomly oriented network of typical amyloid fibrils and more closely resembled fibrils intrinsic to mesangial matrix. The patient had no bone marrow or X-ray evidence of myeloma and no evidence of free monoclonal light chains in serum or concentrated urines. Biosynthetic studies of the patient's bone marrow cells demonstrated unbalanced immunoglobulin synthesis with excess production of monoclonal kappa light chains. These observations suggest that the observed glomerulopathy results from direct deposition of monoclonal light chains. Deposits with kappa light chain determinants have been found in 7 other patients with similar nodular glomerulopathies, 4 of whom had diagnosed clinical myeloma. The lesion of nonamyloidotic nodular glomerulopathy previously described in 19 patients, nor examined by immunopathologic techniques or not shown to contain light chain determinants, may have a similar pathogenesis.

Abnormal heme-protein patterns in hemorrhagic shock.

Allegedly toxic heme pigments have been described in the serum of animals bled to hemorrhagic shock (5,7). In addition, Sears et al. (9), and Braun et al. (1) have shown that the products derived from the degradation of hemoglobin following intravascular hemolysis were toxic (heme carried by hemopexin and albumin). The accumulation of metabolites, caused by the impeded circulation, degrades free hemoglobin into heme pigments and their concentration then reaches a level which exceeds the normal heme-carrying capacity of serum proteins. We have already demonstrated the presence of abnormal heme pigments in clinical cases of shock using scanning spectrophotometry (3). We have endeavored to identify these pigments by serum electrophoresis, and to relate the appearance of some of these compounds to mortality rates. There were no deaths associated with the presence of haptoglobin-hemoglobin alone in serum. As shock deepened and mortality rose, hemopexin-heme and methemalbumin appeared. The highest mortality rate (4 out of 5 cases) was found when both hemopexin-heme and methemalbumin were present. It appears, therefore, that the administration of serum proteins in shock may reduce the toxicity of the products of degradation formed in low-flow states.